The increased prevalence of chronic wounds requires novel treatment options. The aim of this study was to develop a beta-glucan (βG)-loaded nanofiber wound dressing. Nanofibers were prepared using the needle-free Nanospider™ technology, an electrospinning method which enables the production of nanofibers at an industrial scale. The βG was selected as active ingredient based on its confirmed wound healing potential in both animals and humans. Hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO) were included as copolymers. Rheological profiles of spinning solutions containing HPMC, PEO, βG, ethanol and water, were optimized. The nanofiber formation was confirmed by Field Emission Scanning Electron Microscopy (FE-SEM), and both nanofibers with (βG-nanofibers) or without βG (NoβG-nanofibers) were evaluated by their swelling index and FT-IR spectroscopy. The formulations, active ingredient and excipients were tested for their possible in vitro toxicity in keratinocytes. Finally, the wound healing potential of the nanofibers was tested in externally induced excisional wounds in male diabetic db/db mice. Three different doses of βG-nanofibers and the βG-free, NoβG-nanofibers, were evaluated for their in vivo wound healing efficacy. All nanofiber-treatments provided improved wound healing as compared to the negative control (water). All βG-nanofiber treated groups exhibited significantly improved wound healing as compared to the NoβG-nanofiber treated group, indicating the potential of βG-nanofibers as wound dressing.